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Proteintech ube2l6 antibody
Ube2l6 Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ube2l6+antibody/pm41577004-74-1-7?v=Proteintech
Average 94 stars, based on 10 article reviews

ube2l6 antibody - by Bioz Stars, 2026-08

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Downregulation of ISGylation and UBE2L6, but not of UBE1L, Herc5, and USP18 through SPD treatment. ( A ) SPM and SPD partially prevent ISGylation and UBE2L6 expression in MCF10A cells. MCF10A cells are stimulated with hIFN-α with or without SPM or SPD (100, 200, or 400 µM) for 2 days. The cell lysates are subjected to immunoblotting with anti-hISG15, anti-ubiquitin, anti-UBE1L, anti-UBE2L6, anti-Herc5, or anti-USP18 antibody. Ponceau S staining is used as a loading control. Representative data from three independent experiments. ( B ) Quantification of UBE1L, UBE2L6, Herc5, and USP18 expression levels, and that of ISGylation and ubiquitination from ( A ). Each signal is normalized to that of Ponceau S staining. The signal intensity of cells stimulated by human IFN-α is set as 1. Data are expressed as the mean ± standard deviation of three independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as not significant (N.S.), whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively. ( C ) Serum deprivation slows down MCF10A cell growth. MCF10A cells (3 × 10 5 ) are cultured with or without serum in a 6 cm dish, and cell numbers are counted daily. Cell viability is analyzed by trypan blue staining, with stained cells considered dead. ( D ) SPM and SPD partially prevent ISGylation in RAW264.7 cells. RAW264.7 cells are stimulated by LPS with or without SPM or SPD (200, 400, or 800 µM) for 2 days. The cell lysates are subjected to immunoblotting with an anti-mouse ISG15 antibody. Ponceau S staining is used as a loading control. Representative data from five independent experiments. ( E ) Quantification of ISGylation in ( D ). Each signal is normalized to that of Ponceau S staining. The signal intensity of cells stimulated by LPS is set as 1. Data are expressed as the mean ± standard deviation of five independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively.

Journal: Scientific Reports

Article Title: Spermidine reduces ISGylation and enhances ISG15–USP18 interaction

doi: 10.1038/s41598-025-01425-0

Figure Lengend Snippet: Downregulation of ISGylation and UBE2L6, but not of UBE1L, Herc5, and USP18 through SPD treatment. ( A ) SPM and SPD partially prevent ISGylation and UBE2L6 expression in MCF10A cells. MCF10A cells are stimulated with hIFN-α with or without SPM or SPD (100, 200, or 400 µM) for 2 days. The cell lysates are subjected to immunoblotting with anti-hISG15, anti-ubiquitin, anti-UBE1L, anti-UBE2L6, anti-Herc5, or anti-USP18 antibody. Ponceau S staining is used as a loading control. Representative data from three independent experiments. ( B ) Quantification of UBE1L, UBE2L6, Herc5, and USP18 expression levels, and that of ISGylation and ubiquitination from ( A ). Each signal is normalized to that of Ponceau S staining. The signal intensity of cells stimulated by human IFN-α is set as 1. Data are expressed as the mean ± standard deviation of three independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as not significant (N.S.), whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively. ( C ) Serum deprivation slows down MCF10A cell growth. MCF10A cells (3 × 10 5 ) are cultured with or without serum in a 6 cm dish, and cell numbers are counted daily. Cell viability is analyzed by trypan blue staining, with stained cells considered dead. ( D ) SPM and SPD partially prevent ISGylation in RAW264.7 cells. RAW264.7 cells are stimulated by LPS with or without SPM or SPD (200, 400, or 800 µM) for 2 days. The cell lysates are subjected to immunoblotting with an anti-mouse ISG15 antibody. Ponceau S staining is used as a loading control. Representative data from five independent experiments. ( E ) Quantification of ISGylation in ( D ). Each signal is normalized to that of Ponceau S staining. The signal intensity of cells stimulated by LPS is set as 1. Data are expressed as the mean ± standard deviation of five independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively.

Article Snippet: Antibodies against the following proteins were also used: FLAG (1 μg/mL in phosphate-buffered saline [PBS]; M2; Merck), FLAG (used for IP; F2555; Merck), ubiquitin (0.2 μg/mL in Can Get Signal; sc-8017; Santa Cruz Biotechnology), hemagglutinin (0.3 μg/mL in PBS; sc-7392; Santa Cruz Biotechnology), Herc5 (0.5 μg/mL in Can Get Signal; BML-PW0920; Enzo Life Sciences, Farmingdale, NY, USA), UBA1 (0.2 μg/mL in Can Get Signal; sc-53555; Santa Cruz Biotechnology, Dallas, TX, USA), UBE1L (0.2 μg/mL in Can Get Signal; sc-390097; Santa Cruz Biotechnology), UBE2L6 (0.5 μg/mL in Can Get Signal; ab109086; Abcam, Cambridge, MA, USA), Ube2l6 (0.1 μg/mL in Can Get Signal; sc-166276; Santa Cruz Biotechnology), USP18 (1 μg/mL in Can Get Signal; 4813; Cell Signaling Technology, Tokyo, Japan), and V5 (0.2 μg/mL in PBS; sc-271944; Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Ubiquitin Proteomics, Staining, Control, Standard Deviation, Labeling, Cell Culture

Upregulation of ISGylation and the expression of UBE1L, UBE2L6, Herc5, and USP18 through difluoromethylornithine (DFMO) treatment in MCF10A cells. ( A ) DFMO upregulates ISGylation and the expression of UBE1L, UBE2L6, Herc5, and USP18. MCF10A cells are stimulated by hIFN-α with or without DFMO (0.5 or 1.0 mM) for 2 days. The cell lysates are subjected to immunoblotting with anti-ISG15, anti-ubiquitin, anti-UBE1L, anti-UBE2L6, anti-Herc5, or anti-USP18 antibody. Ponceau S staining is used as a loading control. Representative data from five independent experiments. ( B ) Quantification of UBE1L, UBE2L6, Herc5, USP18, and free ISG15 expression levels, as well as ISGylation and ubiquitination from ( A ). Each signal is normalized to Ponceau S staining. The signal intensity of cells stimulated by human IFN-α is set to 1. Data are expressed as the mean ± standard deviation of five independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively. ( C ) Upregulation of ISGylation by DFMO in the absence of UBA1. UBA1-knockdown or control MCF10A cells are stimulated with hIFN-α with or without DFMO (0.5 mM) for 2 days. The cell lysates are subjected to immunoblotting using anti-UBA1, anti-ISG15, and anti-ubiquitin antibodies. Ponceau S staining is used as a loading control. Representative data from three independent experiments. ( D ) Quantification of UBA1 expression levels, ISGylation, and ubiquitination from ( C ). Each signal is normalized to Ponceau S staining. The signal intensity of UBA1 and ubiquitination in control knockdown cells stimulated by hIFN-α is set to 1. In contrast, the signal intensity of ISGylation in control and UBA1-knockdown cells stimulated by hIFN-α is set to 1. Data are expressed as the mean ± standard deviation of three independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively.

Journal: Scientific Reports

Article Title: Spermidine reduces ISGylation and enhances ISG15–USP18 interaction

doi: 10.1038/s41598-025-01425-0

Figure Lengend Snippet: Upregulation of ISGylation and the expression of UBE1L, UBE2L6, Herc5, and USP18 through difluoromethylornithine (DFMO) treatment in MCF10A cells. ( A ) DFMO upregulates ISGylation and the expression of UBE1L, UBE2L6, Herc5, and USP18. MCF10A cells are stimulated by hIFN-α with or without DFMO (0.5 or 1.0 mM) for 2 days. The cell lysates are subjected to immunoblotting with anti-ISG15, anti-ubiquitin, anti-UBE1L, anti-UBE2L6, anti-Herc5, or anti-USP18 antibody. Ponceau S staining is used as a loading control. Representative data from five independent experiments. ( B ) Quantification of UBE1L, UBE2L6, Herc5, USP18, and free ISG15 expression levels, as well as ISGylation and ubiquitination from ( A ). Each signal is normalized to Ponceau S staining. The signal intensity of cells stimulated by human IFN-α is set to 1. Data are expressed as the mean ± standard deviation of five independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively. ( C ) Upregulation of ISGylation by DFMO in the absence of UBA1. UBA1-knockdown or control MCF10A cells are stimulated with hIFN-α with or without DFMO (0.5 mM) for 2 days. The cell lysates are subjected to immunoblotting using anti-UBA1, anti-ISG15, and anti-ubiquitin antibodies. Ponceau S staining is used as a loading control. Representative data from three independent experiments. ( D ) Quantification of UBA1 expression levels, ISGylation, and ubiquitination from ( C ). Each signal is normalized to Ponceau S staining. The signal intensity of UBA1 and ubiquitination in control knockdown cells stimulated by hIFN-α is set to 1. In contrast, the signal intensity of ISGylation in control and UBA1-knockdown cells stimulated by hIFN-α is set to 1. Data are expressed as the mean ± standard deviation of three independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively.

Techniques: Expressing, Western Blot, Ubiquitin Proteomics, Staining, Control, Standard Deviation, Labeling, Knockdown

Upregulation of ISGylation through DFMO treatment and downregulation of ISGylation through SPD treatment in the presence of DFMO in A549 cells. ( A ) Serum deprivation slows down A549 cell growth. A549 cells (2 × 10 5 ) are cultured with or without serum in a 6 cm dish, and cell numbers are counted daily. Cell viability is analyzed by trypan blue staining, with stained cells considered dead. ( B ) DFMO slightly upregulates ISGylation but not ubiquitination. MCF10A cells are stimulated by hIFN-α with or without DFMO (0.5 or 1.0 mM) for 2 days. The cell lysates are subjected to immunoblotting using an anti-ISG15, anti-ubiquitin, anti-UBE1L, anti-UBE2L6, anti-Herc5, or anti-USP18 antibody. Ponceau S staining is used as a loading control. Representative data from five independent experiments. ( C ) Quantification of UBE1L, UBE2L6, Herc5, and USP18 expression levels, as well as that of ISGylation and ubiquitination in ( B ). Each signal is normalized to that of Ponceau S staining. The signal intensity of cells stimulated by human IFN-α is set as 1. Data are expressed as the mean ± standard deviation of five independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively. ( D ) SPD partially prevents ISGylation, ubiquitination, UBE1L, UBE2L6, and USP18 expression in the A549 cells. A549 cells are stimulated by hIFN-α with or without SPD (0.5 or 1.0 mM) in the presence of DFMO (1.0 mM) for 2 days. The cell lysates are subjected to immunoblotting with anti-ISG15, anti-ubiquitin, anti-UBE1L, anti-UBE2L6, anti-Herc5, or anti-USP18 antibody. Ponceau S staining is used as a loading control. Representative data of three independent experiments. ( E ) Quantification of UBE1L, UBE2L6, Herc5, and USP18 expression levels, and that of ISGylation and ubiquitination in ( D ). Each signal is normalized to that of Ponceau S staining. The signal intensity of cells stimulated by human IFN-α is set as 1. Data are expressed as the mean ± standard deviation of three independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively.

Journal: Scientific Reports

Article Title: Spermidine reduces ISGylation and enhances ISG15–USP18 interaction

doi: 10.1038/s41598-025-01425-0

Figure Lengend Snippet: Upregulation of ISGylation through DFMO treatment and downregulation of ISGylation through SPD treatment in the presence of DFMO in A549 cells. ( A ) Serum deprivation slows down A549 cell growth. A549 cells (2 × 10 5 ) are cultured with or without serum in a 6 cm dish, and cell numbers are counted daily. Cell viability is analyzed by trypan blue staining, with stained cells considered dead. ( B ) DFMO slightly upregulates ISGylation but not ubiquitination. MCF10A cells are stimulated by hIFN-α with or without DFMO (0.5 or 1.0 mM) for 2 days. The cell lysates are subjected to immunoblotting using an anti-ISG15, anti-ubiquitin, anti-UBE1L, anti-UBE2L6, anti-Herc5, or anti-USP18 antibody. Ponceau S staining is used as a loading control. Representative data from five independent experiments. ( C ) Quantification of UBE1L, UBE2L6, Herc5, and USP18 expression levels, as well as that of ISGylation and ubiquitination in ( B ). Each signal is normalized to that of Ponceau S staining. The signal intensity of cells stimulated by human IFN-α is set as 1. Data are expressed as the mean ± standard deviation of five independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively. ( D ) SPD partially prevents ISGylation, ubiquitination, UBE1L, UBE2L6, and USP18 expression in the A549 cells. A549 cells are stimulated by hIFN-α with or without SPD (0.5 or 1.0 mM) in the presence of DFMO (1.0 mM) for 2 days. The cell lysates are subjected to immunoblotting with anti-ISG15, anti-ubiquitin, anti-UBE1L, anti-UBE2L6, anti-Herc5, or anti-USP18 antibody. Ponceau S staining is used as a loading control. Representative data of three independent experiments. ( E ) Quantification of UBE1L, UBE2L6, Herc5, and USP18 expression levels, and that of ISGylation and ubiquitination in ( D ). Each signal is normalized to that of Ponceau S staining. The signal intensity of cells stimulated by human IFN-α is set as 1. Data are expressed as the mean ± standard deviation of three independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively.

Techniques: Cell Culture, Staining, Ubiquitin Proteomics, Western Blot, Control, Expressing, Standard Deviation, Labeling

D103A or E133L mutations in mISG15 enhanced ISGylation in the presence of SPD. ( A ) UBE1L, Ube2l6, HA-Herc5, and FLAG-mISG15 (wild type [WT] or mutant) are expressed in the HEK293T cells in the presence of SPD. 2 days after transfection, the cell lysates are subjected to immunoblotting using anti-FLAG, anti-UBE1L, anti-Ube2l6, and anti-HA antibodies. Ponceau S staining is used as a loading control. Representative data of three independent experiments. ( B ) Quantification of FLAG-mISGylation, and UBE1L, Ube2l6, and HA-Herc5 expression levels in ( A ). Each signal is normalized to that of Ponceau S staining. The signal intensity of cells expressing FLAG-mISG15 (WT) and ISGylation system is set as 1. Data are expressed as the mean ± standard deviation of three independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively.

Journal: Scientific Reports

Article Title: Spermidine reduces ISGylation and enhances ISG15–USP18 interaction

doi: 10.1038/s41598-025-01425-0

Figure Lengend Snippet: D103A or E133L mutations in mISG15 enhanced ISGylation in the presence of SPD. ( A ) UBE1L, Ube2l6, HA-Herc5, and FLAG-mISG15 (wild type [WT] or mutant) are expressed in the HEK293T cells in the presence of SPD. 2 days after transfection, the cell lysates are subjected to immunoblotting using anti-FLAG, anti-UBE1L, anti-Ube2l6, and anti-HA antibodies. Ponceau S staining is used as a loading control. Representative data of three independent experiments. ( B ) Quantification of FLAG-mISGylation, and UBE1L, Ube2l6, and HA-Herc5 expression levels in ( A ). Each signal is normalized to that of Ponceau S staining. The signal intensity of cells expressing FLAG-mISG15 (WT) and ISGylation system is set as 1. Data are expressed as the mean ± standard deviation of three independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively.

Techniques: Mutagenesis, Transfection, Western Blot, Staining, Control, Expressing, Standard Deviation, Labeling

SPD enhances ISGylation independent of ISG15 activation by UBE1L in the HEK293T cell. ( A ) SPD enhances ectopically-induced ISGylation in the HEK293T cells. UBE1L, Ube2l6, HA-Herc5, and FLAG- mISG15 are expressed in the HEK293T cell with or without SPD (0.5 or 1.5 mM). 2 days after transfection, cell lysates are subjected to immunoblotting with anti-FLAG, anti-UBE1L, anti-Ube2l6, and anti-HA antibody. Ponceau S staining is used as a loading control. Representative data from four independent experiments. ( B ) Quantification of FLAG-mISGylation, and UBE1L, Ube2l6, and HA-Herc5 expression levels in ( A ). Each signal is normalized to that of Ponceau S staining. The signal intensity of cells expressing FLAG-mISG15 without SPD is set to 1. Data are expressed as the mean ± standard deviation of four independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively. ( C ) SPD did not affect the activation of ISG15. UBE1L and FLAG-mISG15 are expressed in the HEK293T cells with or without SPD (0.5 or 1.5 mM) in the presence of DFMO (1 mM). 2 days after transfection, cell lysates are subjected to immunoblotting using an anti-FLAG or anti-UBE1L antibody. Ponceau S staining is used as a loading control. Representative data of four independent experiments. ( D ) Quantification of FLAG-mISG15-UBE1L in ( C ). The signal intensity of FLAG-mISG15-UBE1L without SPD is set to 1. Data are expressed as the mean ± standard deviation of four independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively.

Journal: Scientific Reports

Article Title: Spermidine reduces ISGylation and enhances ISG15–USP18 interaction

doi: 10.1038/s41598-025-01425-0

Figure Lengend Snippet: SPD enhances ISGylation independent of ISG15 activation by UBE1L in the HEK293T cell. ( A ) SPD enhances ectopically-induced ISGylation in the HEK293T cells. UBE1L, Ube2l6, HA-Herc5, and FLAG- mISG15 are expressed in the HEK293T cell with or without SPD (0.5 or 1.5 mM). 2 days after transfection, cell lysates are subjected to immunoblotting with anti-FLAG, anti-UBE1L, anti-Ube2l6, and anti-HA antibody. Ponceau S staining is used as a loading control. Representative data from four independent experiments. ( B ) Quantification of FLAG-mISGylation, and UBE1L, Ube2l6, and HA-Herc5 expression levels in ( A ). Each signal is normalized to that of Ponceau S staining. The signal intensity of cells expressing FLAG-mISG15 without SPD is set to 1. Data are expressed as the mean ± standard deviation of four independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively. ( C ) SPD did not affect the activation of ISG15. UBE1L and FLAG-mISG15 are expressed in the HEK293T cells with or without SPD (0.5 or 1.5 mM) in the presence of DFMO (1 mM). 2 days after transfection, cell lysates are subjected to immunoblotting using an anti-FLAG or anti-UBE1L antibody. Ponceau S staining is used as a loading control. Representative data of four independent experiments. ( D ) Quantification of FLAG-mISG15-UBE1L in ( C ). The signal intensity of FLAG-mISG15-UBE1L without SPD is set to 1. Data are expressed as the mean ± standard deviation of four independent experiments. The means of different groups are compared using the Student’s t -test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively.

Techniques: Activation Assay, Transfection, Western Blot, Staining, Control, Expressing, Standard Deviation, Labeling

Fig. 4 Decursin decreases the FLT3-ITD protein level. A Volcano plots were analyzed for the bases with more than 1.2-fold upregulation (red plot) and downregulation (green plot) of decursin. B Heatmap of differential proteins between treatment group and control group. C Significantly increased proteins following decursin treatment. D GSEA of proteomic data from MV4-11 cells treated with decursin before and after delivery. The figure shows the GSEA enrichment plot of the relevant proteins in the ubiquitin‒proteasome pathway after decursin treatment (n = 2). E–G Expression of C-MYC, UBE2L6, and PIM1, as well as total and phosphorylated FLT3, STAT5, AKT, and ERK was assessed. (n = 3)

Journal: Cell communication and signaling : CCS

Article Title: Decursin induces FLT3-ITD acute myeloid leukemia cell apoptosis by increasing the expression of the ubiquitin-conjugase UBE2L6.

doi: 10.1186/s12964-025-02157-4

Figure Lengend Snippet: Fig. 4 Decursin decreases the FLT3-ITD protein level. A Volcano plots were analyzed for the bases with more than 1.2-fold upregulation (red plot) and downregulation (green plot) of decursin. B Heatmap of differential proteins between treatment group and control group. C Significantly increased proteins following decursin treatment. D GSEA of proteomic data from MV4-11 cells treated with decursin before and after delivery. The figure shows the GSEA enrichment plot of the relevant proteins in the ubiquitin‒proteasome pathway after decursin treatment (n = 2). E–G Expression of C-MYC, UBE2L6, and PIM1, as well as total and phosphorylated FLT3, STAT5, AKT, and ERK was assessed. (n = 3)

Article Snippet: Antibodies against GAPDH (81640–1-RR), cyclin E2 (11935–1-AP), Lamin B1 (66095–1-Ig), PARP1 (13371–1-AP), β-actin (81115–1-RR), STAT5 (80138–2-RR), MCL-1 (16225–1-AP), AKT (10176– 2-AP), ERK1/2 (83533–1-RR), UBE2L6 (17278–1-AP), SIAH1 (83389–4-RR), and CDK4 (11026–1-AP), as well as HRP-conjugated IgG antibodies (SA00001), were from ProteinTech (Wuhan, China).

Techniques: Control, Expressing

Fig. 6 Decursin-induced apoptosis occurs due to an increase in UBE2L6. A mRNA levels of USP10, USP9X, UBE2L6, and SIAH1 relative to those of GAPDH. B After treatment with decursin, the levels of UBE2L6 and SIAH1 were evaluated using Western blotting. C The levels of UBE2L6 in FLT3-positive (n = 50) and FLT3-negative (n = 116) AML patients from the TCGA database were analyzed using UALCAN (https://ualcan.path.uab. edu/index.html). LAML: TCGA project code for acute myeloid leukemia (AML). D Expression of UBE2L6 was tested. E MV4-11 and MOLM-13 were transfected with a lentiviral vector carrying a negative control or containing three small hairpin RNAs (shRNAs) for UBE2L6. F-I Colony formation rates and apoptosis rates were assessed in the negative control or UBE2L6-knockdown groups treated with decursin. J, K Expression of C-MYC and UBE2L6, as well as total and phosphorylated FLT3 and STAT5, in the negative control or UBE2L6-knockdown groups treated with decursin. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001)

Journal: Cell communication and signaling : CCS

Article Title: Decursin induces FLT3-ITD acute myeloid leukemia cell apoptosis by increasing the expression of the ubiquitin-conjugase UBE2L6.

doi: 10.1186/s12964-025-02157-4

Figure Lengend Snippet: Fig. 6 Decursin-induced apoptosis occurs due to an increase in UBE2L6. A mRNA levels of USP10, USP9X, UBE2L6, and SIAH1 relative to those of GAPDH. B After treatment with decursin, the levels of UBE2L6 and SIAH1 were evaluated using Western blotting. C The levels of UBE2L6 in FLT3-positive (n = 50) and FLT3-negative (n = 116) AML patients from the TCGA database were analyzed using UALCAN (https://ualcan.path.uab. edu/index.html). LAML: TCGA project code for acute myeloid leukemia (AML). D Expression of UBE2L6 was tested. E MV4-11 and MOLM-13 were transfected with a lentiviral vector carrying a negative control or containing three small hairpin RNAs (shRNAs) for UBE2L6. F-I Colony formation rates and apoptosis rates were assessed in the negative control or UBE2L6-knockdown groups treated with decursin. J, K Expression of C-MYC and UBE2L6, as well as total and phosphorylated FLT3 and STAT5, in the negative control or UBE2L6-knockdown groups treated with decursin. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001)

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Negative Control, Knockdown

Fig. 8 Effects of decursin in primary AML cells. A-C Apoptosis rates induced by decursin treatment for 24 h in patient and healthy human samples. D Expression of UBE2L6 mRNA levels were assessed. E Total and phosphorylated FLT3, STAT5, AKT, and ERK, as well as UBE2L6 in FLT3-ITD primary cells were assessed. F Schematic diagram of the PDX model. G CD45-positive rates were quantitatively detected (n = 3). H The Kaplan‒Meier method was utilized to assess animal survival. (n = 5) (*P < 0.05, **P < 0.01)

Journal: Cell communication and signaling : CCS

Article Title: Decursin induces FLT3-ITD acute myeloid leukemia cell apoptosis by increasing the expression of the ubiquitin-conjugase UBE2L6.

doi: 10.1186/s12964-025-02157-4

Figure Lengend Snippet: Fig. 8 Effects of decursin in primary AML cells. A-C Apoptosis rates induced by decursin treatment for 24 h in patient and healthy human samples. D Expression of UBE2L6 mRNA levels were assessed. E Total and phosphorylated FLT3, STAT5, AKT, and ERK, as well as UBE2L6 in FLT3-ITD primary cells were assessed. F Schematic diagram of the PDX model. G CD45-positive rates were quantitatively detected (n = 3). H The Kaplan‒Meier method was utilized to assess animal survival. (n = 5) (*P < 0.05, **P < 0.01)

Techniques: Expressing

a , HEK293-CD13-myc ISG15 stable cells were infected with HCoV-229E at an MOI of 0.01 for 72 h prior to collection of cell lysates (lysates) and supernatant (S/N) prior to western blot with anti-myc Ab (as above) to detect transfected ISG15. The level of ISG15 in the supernatant was normalized to cell lysates and loading control (GAPDH), and was expressed relative to the most abundant protein in the supernatant ( Mops condylurus ). Only ISG15 of Mops condylurus was significantly secreted into the supernatant and secretion further increased during HCoV-229E infection. Mean and standard error of the mean are displayed, including individual points (black) for n = 3 independent experiments. Significance was tested with a two-tailed t-test comparing infection to ISG15 alone. Raw data are provided in Supplementary Table . b , An example western blot image (as per panel a) showing high and low exposure of ISG15 supernatant (S/N), HCoV-229E N protein, GAPDH and ISG15 bands in the HEK293-subclone-ΔISG15 cell lysate with protein ladders. Lanes are as indicated. c , Example western blot image with low and high contrast showing ISG15 conjugation (α-myc) after transfection with ISG15’s and HCoV-229E infection (−/+). Arrows indicate bands not seen in the empty vector control (conjugated proteins). d , Western blot of E1 ligase (UBE1L), E2 ligase (UBE2L6) and E3 ligase (HERC5) expression, together with GAPDH in the same samples shown in panel c, indicating sufficient expression of ISGylation machinery in the HEK293-subclone-ΔISG15 cell line. e , HEK293-subclone-ΔISG15 cells were transfected with ISG15 constructs as indicated and with/without NSP3C/L (SARS-CoV-2 PLpro). As per panel c, arrows indicate ISGylation bands, or ISG15 dimer/monomer. The amount of ISG15 present was calculated, normalized to GAPDH and quantified from three western blots. One of the three western blots is shown as an example. f , Representative western blot for the amount of ISG15 present in the supernatant (matched to e). g , ISGylation machinery expression, as per panel d, for NSP3C samples used in panel e and f and Fig. . Western blots in panels b-g are matched to the quantification in Fig. .

Journal: Nature

Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance

doi: 10.1038/s41586-024-08471-0

Figure Lengend Snippet: a , HEK293-CD13-myc ISG15 stable cells were infected with HCoV-229E at an MOI of 0.01 for 72 h prior to collection of cell lysates (lysates) and supernatant (S/N) prior to western blot with anti-myc Ab (as above) to detect transfected ISG15. The level of ISG15 in the supernatant was normalized to cell lysates and loading control (GAPDH), and was expressed relative to the most abundant protein in the supernatant ( Mops condylurus ). Only ISG15 of Mops condylurus was significantly secreted into the supernatant and secretion further increased during HCoV-229E infection. Mean and standard error of the mean are displayed, including individual points (black) for n = 3 independent experiments. Significance was tested with a two-tailed t-test comparing infection to ISG15 alone. Raw data are provided in Supplementary Table . b , An example western blot image (as per panel a) showing high and low exposure of ISG15 supernatant (S/N), HCoV-229E N protein, GAPDH and ISG15 bands in the HEK293-subclone-ΔISG15 cell lysate with protein ladders. Lanes are as indicated. c , Example western blot image with low and high contrast showing ISG15 conjugation (α-myc) after transfection with ISG15’s and HCoV-229E infection (−/+). Arrows indicate bands not seen in the empty vector control (conjugated proteins). d , Western blot of E1 ligase (UBE1L), E2 ligase (UBE2L6) and E3 ligase (HERC5) expression, together with GAPDH in the same samples shown in panel c, indicating sufficient expression of ISGylation machinery in the HEK293-subclone-ΔISG15 cell line. e , HEK293-subclone-ΔISG15 cells were transfected with ISG15 constructs as indicated and with/without NSP3C/L (SARS-CoV-2 PLpro). As per panel c, arrows indicate ISGylation bands, or ISG15 dimer/monomer. The amount of ISG15 present was calculated, normalized to GAPDH and quantified from three western blots. One of the three western blots is shown as an example. f , Representative western blot for the amount of ISG15 present in the supernatant (matched to e). g , ISGylation machinery expression, as per panel d, for NSP3C samples used in panel e and f and Fig. . Western blots in panels b-g are matched to the quantification in Fig. .

Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).

Techniques: Infection, Western Blot, Transfection, Control, Two Tailed Test, Conjugation Assay, Plasmid Preparation, Expressing, Construct

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